Movements of PSD components during activity were studied by pre-embedding Nanogold labeling of dissociated hippocampal neurons in culture. Depolarization with high K+ is typically used to depolarize and activate neuronal cultures, but other protocols closer to physiological stimulation, such as application of NMDA and synaptic activation with glycine in the absence of Mg2+, are also used. Now that antibodies for nearly all major components of the PSD have been obtained and tested for compatibility for immunoelectron microscopy, a general picture of activity-induced reorganization of the PSD at the molecular level begins to emerge. Scaffold proteins such as PSD-95 and GKAP appear to be relatively stable during short-term activity, and constitute a layer 30 nm thick lying next to the post synaptic membrane where receptors are stabilized by associations with the scaffold. &#8232;&#8232;In contrast, other proteins change location during short-term activity. For instance, Shank2 moves closer to the PSD after stimulation. While we have previously described movement of CaMKII towards the PSD during activation, we have now showed that another major regulatory protein, SynGAP, moves away from the PSD upon stimulation. The changes induced by stimulation coincide with a marked increase of AMPA receptor labeling at the PSD, compatible with enzymatic as well as structural roles of CaMKII and SynGAP in regulating AMPA receptor trafficking at the PSD. The mechanism of activity-induced SynGAP translocation is being investigated. SynGAP is known to bind to PSD-95 and this association may be the anchor that localizes it to the PSD core. Incubation of isolated PSDs under conditions that promote CaMKII activation causes phosphorylation of SynGAP and disrupts its co-immunopreciptation with PSD-95. CYLD is another protein that moves toward the PSD upon activation. CYLD is a deubiquitinase specific for Lys-63 linked polyubiquitins. CYLD is highly enriched in PSD fractions as seen by western blotting and mass spectrometry. Immunogold EM shows localizes CYLD in cell bodies and dendrites, but not in axons, and few PSDs in cultured hippocampal neurons label for CYLD under basal conditions. Depolarization promotes further accumulation of CYLD at PSDs, doubling the number of synapses labeled for CYLD as well as increasing the intensity of CYLD labeling. Increased CYLD activity at the synapse could prevent lysosomal degradation of synaptic proteins and regulate their intracellular trafficking. Enrichment of CYLD at the PSD has now been confirmed with two antibodies raised to non- overlapping sequences of CYLD. The increase in immunogold labeling for CYLD upon activity has been confirmed and further documented. New studies using CYLD KO mice, a collaboration with Ashish Jain, NIAID, compare levels and patterns of protein ubiquitination and amounts of aggregated/detergent insoluble proteins with wild type mice,&#8232;&#8232; EM tomography of isolated PSDs is being used to describe with more precision the re-localization of proteins at the PSD following activity. Two technical breakthroughs, the isolation of PSD fractions from hippocampal slice cultures, and a negative staining method compatible with EM tomography, lie behind this new work. PSDs isolated from depolarized hippocampal slices show elevated levels of CaMKII confirming that activity-induced changes at the PSD are preserved during isolation. CaMKII association domains are readily recognizable in negative stain tomograms, allowing accurate localization of individual CaMKII molecules within the PSD. We plan to determine exactly where in the PSD CaMKII is bound after stimulation protocols, such as such as chemLTP and chemLTD, because CaMKII has multiple phosphorylation targets at multiple locations within the PSD. Quantitative mass spectrometry collaborations are in place to track parallel changes in protein composition and phosphorylation.